Fab I inhibitors

ABSTRACT

Compounds of the formula (I) are disclosed which are FabI inhibitors and are useful in the treatment of bacterial infections.

This application claims the benefit of Provisional application No.60/258,529, filed Oct. 8, 1999.

FIELD OF THE INVENTION

This invention relates to pharmaceutically active compounds whichinhibit FabI and are useful for the treatment of bacterial infections.

BACKGROUND OF THE INVENTION

While the overall pathway of saturated fatty acid biosynthesis issimilar in all organisms, the fatty acid synthase (FAS) systems varyconsiderably with respect to their structural organization. Vertebratesand yeast possess a FAS in which all the enzymatic activities areencoded on one or two polypeptide chains, respectively, and the acylcarrier protein (ACP) is an integral part of the complex. In contrast,in bacterial FAS, each of the reactions is catalyzed by a distinct,mono-functional enzyme and the ACP is a discrete protein. Therefore,there is considerable potential for the selective inhibition of thebacterial system by antibacterial agents.

FabI (previously designated EnvM) functions as an enoyl-ACP reductase(Bergler, et al, (1994), J. Biol. Chem. 269, 5493-5496) in the finalstep of the four reactions involved in each cycle of bacterial fattyacid biosynthesis. In this pathway, the first step is catalyzed byβ-ketoacyl-ACP synthase, which condenses malonyl-ACP with acetyl-CoA(FabH, synthase III). In subsequent rounds, malonyl-ACP is condensedwith the growing-chain acyl-ACP (FabB and FabF, synthases I and II,respectively). The second step in the elongation cycle is ketoesterreduction by NADPH-dependent β-ketoacyl-ACP reductase (FabG). Subsequentdehydration by β-hydroxyacyl-ACP dehydrase (either FabA or FabZ) leadsto trans-2-enoyl-ACP, which in turn is converted to acyl-ACP byNADH-dependent enoyl-ACP reductase (FabI). Further rounds of this cycle,adding two carbon atoms per cycle, eventually lead to palmitoyl-ACP(16C), where upon the cycle is stopped largely due to feedbackinhibition of FabI by palmitoyl-ACP (Heath, et al, (1996), J. Biol.Chem. 271, 1833-1836). Thus, FabI is a major biosynthetic enzyme and isa key regulatory point in the overall synthetic pathway of bacterialfatty acid biosynthesis. Therefore, FabI is an ideal target forantibacterial intervention.

Studies have shown that diazaborine antibiotics inhibit fatty acid,phospholipid and lipopolysaccharide (LPS) biosynthesis and that theantibacterial target of these compounds is FabI. For example, derivative2b18 from Grassberger, et al (1984) J. Med Chem 27 947-953 has beenreported to be a non-competitive inhibitor of FabI (Bergler, et al,(1994), J. Biol. Chem. 269. 5493-5496). Also, plasmids containing theFabI gene from diazaborine resistant S. typhimurium conferreddiazaborine resistance in E. coli (Turnowsky, et al, 1989), J.Bacteriol., 171, 6555-6565). Furthermore, inhibition of FabI either bydiazaborine or by raising the temperature in a FabI temperaturesensitive mutant is lethal. these results demonstrate that FabI isessential to the survival of the organism (Bergler, et al, (1994), J.Biol. Chem. 269, 5493-5496).

Recent studies have shown that FabI is also the target for the broadspectrum antibacterial agent triclosan (McMurry, et al, (1998) Nature394, 531-532). A crystal structure of the E. coli FabI complexed withNAD and triclosan shows that triclosan acts as a site-directed, verypotent inhibitor of FabI by mimicking its natural substrate (Levy, etal, (1999) Nature 398, 383-384). Ward, et al ((1999) Biochem. 38,12514-12525) have shown that there is no evidence for the formation of acovalent complex between FabI and triclosan, which would be analogous tothe diazaborines; triclosan differs from these compounds in that it is areversible inhibitor of FabI. The structural data for the complex ofFabI with NAD and triclosan provides important information about FabI asa therapeutic target.

Importantly, it has now been discovered that certain compounds are FabIinhibitors and have antibacterial activity, and, therefore, may beuseful for the treatment of bacterial infections in mammals,particularly in man.

SUMMARY OF THE INVENTION

This invention comprises compounds of the formula (I), as describedhereinafter, which inhibit FabI and are useful in the treatment ofbacterial infections.

This invention is also a pharmaceutical composition comprising acompound according to formula (I) and a pharmaceutically acceptablecarrier.

This invention is also a method of treating bacterial infections byinhibiting FabI. In a particular aspect, the compounds of this inventionare useful as antibacterial agents.

DETAILED DESCRIPTION

This invention comprises compounds of formula (I):

wherein:

R¹ is C₁₋₄alkyl;

R² is C₁₋₄alkyl;

R³ is —C₁₋₄alkyl, —C₀₋₄alkyl-Ar or —C₀₋₄alkyl-Het;

R⁴ is —C₁₋₄alkyl, —(CH₂)₁₋₄OH, —OC₁₋₄alkyl, —SC₁₋₄alkyl, —N(C₁₋₄alkyl)₂,—C₀₋₄alkyl-Ar, —C₀₋₄alkyl-Het, —C₀₋₄alkyl-C₃₋₆cycloalkyl, —CH(OH)—CH₂—R*or —(CH₂)₁₋₃SO₂Ar;

R* is C₁₋₄alkyl, Ar or Het:

X is H, C₁₋₄alkyl, OR′, SR′, CN, N(R′)₂, CH₂N(R′)₂, NO₂, CF₃, CO₂R′,CON(R′)₂, COR′, NR′C(O)R′, F, Cl, Br, I, or —S(O)_(r)CF₃;

R′ is H, C₁₋₆alkyl or —C₀₋₆alkyl-Ar; and

r is 0, 1 or 2;

or a pharmaceutically acceptable salt thereof.

Also included in this invention are pharmaceutically acceptable additionsalts and complexes of the compounds of this invention. In cases whereinthe compounds of this invention may have one or more chiral centers,unless specified, this invention includes each unique racemic compound,as well as each unique nonracemic compound.

In cases in which compounds have unsaturated carbon-carbon double bonds,both the cis (Z) and trans (E) isomers are within the scope of thisinvention. In cases wherein compounds may exist in tautomeric forms,such as keto-enol tautomers, such as

each tautomeric form is contemplated as being included within thisinvention, whether existing in equilibrium or locked in one form byappropriate substitution with R′. The meaning of any substituent at anyone occurrence is independent of its meaning, or any other substituent'smeaning, at any other occurrence.

Also included in this invention are prodrugs of the compounds of thisinvention. Prodrugs are considered to be any covalently bonded carrierswhich release the active parent drug according to formula (I) in vivo.

The compounds of formula (I) inhibit FabI. Inhibition of this enzyme isuseful in the treatment of bacterial infections. Also, the compounds ofthis invention may be useful as antifungal agents. Additionally, thecompounds may be useful in combination with known antibiotics.

With respect to formula (I), this invention preferably includescompounds of formula (Ia):

in which R³ and R⁴ are as defined for formula (I) compounds.

Suitably, with respect to formula (I), R³ is —C₁₋₄alkyl or —C₀₋₂alkyl-Phand R⁴ is —C₁₋₄alkyl, —CH₂OH, —OC₁₋₄alkyl, —C₀₋₂alkyl-Ph,—C₀₋₂alkyl-C₃₋₆cycloalkyl, —CH(OH)—CH₂—R* or —(CH₂)₂SO₂Ph, in which R*is as defined for formula (I) compounds.

Representative of the novel compounds of this invention are thefollowing:

N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-methylacetamide;

N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-(2-phenylethyl)acetamide;

N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-2-hydroxy-4-methyl-N-methylpentanamide;

{4-amino-3-[(ethoxy-N-methylcarbonylamino)methyl]phenyl}-N-methyl-N-[(1-methylindol-2-yl)methyl]carboxamide;

N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-2-hydroxy-N-methylacetamide:

N-[(2-amino-5-{N-methyl-N-[(1-methylindol-3-yl)methyl]carbamoyl}phenyl)methyl]-N-methylacetamide,

N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-phenylacetamide;

N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-2-hydroxy-3-indol-3-yl-N-methylpropanamide;

(4-amino-3-{[(4-hydroxyphenyl)-N-methylcarbonylamino]methyl}phenyl)-N-methyl-N-[(1-methylindol-2-yl)methyl]carboxamide;

N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-methyl-3-(phenylsulfonyl)propanamide;and

N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-2-cyclopentyl-N-methylacetamide;

or a pharmaceutically acceptable salt thereof.

Abbreviations and symbols commonly used in the peptide and chemical artsare used herein to describe the compounds of this invention. In general,the amino acid abbreviations follow the IUPAC-IUB Joint Commission onBiochemical Nomenclature as described in Eur. J. Biochem., 158, 9(1984).

C₁₋₄alkyl as applied herein means an optionally substituted alkyl groupof 1 to 4 carbon atoms, and includes methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl and t-butyl, C₁₋₆alkyl additionally includes pentyl,n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphaticisomers thereof, C₀₋₄alkyl and C₀₋₆alkyl additionally indicates that noalkyl group need be present (e.g., that a covalent bond is present).

Any C₁₋₄alkyl or C₁₋₆ alkyl may be optionally substituted with the groupR^(X), which may be on any carbon atom that results in a stablestructure and is available by conventional synthetic techniques.Suitable groups for R^(X) are C₁₋₄alkyl, OR′, SR′, CN, N(R′)₂,CH₂N(R′)₂, —NO₂, —CF₃, —CO₂R′—CON(R′)₂, —COR′, —NR′C(O)R′, F, Cl, Br, I,or —S(O)_(r)CF₃, wherein R′ and r are as defined for formula (I)compounds.

Halogen or halo means F, Cl, Br, and I.

Ar, or aryl, as applied herein, means phenyl or naphthyl, or phenyl ornaphthyl substituted by one to three substituents, such as those definedabove for alkyl, or substituted by methylenedioxy.

Het, or heterocycle, indicates an optionally substituted five or sixmembered monocyclic ring, or a nine or ten-membered bicyclic ringcontaining one to three heteroatoms chosen from the group of nitrogen,oxygen and sulfur, which are stable and available by conventionalchemical synthesis. Illustrative heterocycles are benzofuryl,benzimidazolyl, benzopyranyl, benzothienyl, furyl, imidazolyl,indolinyl, morpholinyl, piperidinyl, piperazinyl, pyrrolyl,pyrrolidinyl, tetrahydropyridinyl, pyridinyl, thiazolyl, thienyl,quinolinyl, isoquinolinyl, and tetra- and perhydro-quinolinyl andisoquinolinyl. Any accessible combination of up to three substituents onthe Het ring, such as those defined above for alkyl, that are availableby chemical synthesis and are stable are within the scope of thisinvention.

Certain radical groups are abbreviated herein. t-Bu refers to thetertiary butyl radical, Boc refers to the t-butyloxycarbonyl radical.Fmoc refers to the fluorenylmethoxycarbonyl radical, Ph refers to thephenyl radical, Cbz refers to the benzyloxycarbonyl radical. Bn refersto the benzyl radical, Me refers to methyl, Et refers to ethyl, Acrefers to acetyl, Alk refers to C₁₋₄alkyl, Nph refers to 1- or2-naphthyl and cHex refers to cyclohexyl. Tet refers to 5-tetrazolyl.

Certain reagents are abbreviated herein. DCC refers todicyclohexylcarbodiimide. DMAP refers to dimethylaminopyridine, EDCrefers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride,HOBt refers to 1-hydroxybenzotriazole, THF refers to tetrahydrofuran,DIEA refers to diisopropylethylamine, DEAD refers to diethylazodicarboxylate, PPh₃ refers to triphenylphosphine, DIAD refers todiisopropyl azodicarboxylate, DME refers to dimethoxyethane, DMF refersto dimethylformamide, NBS refers to N-bromosuccinimide, Pd/C refers to apalladium on carbon catalyst, PPA refers to polyphosphoric acid, DPPArefers to diphenylphosphoryl azide, BOP refers tobenzotriazol-1-yloxy-tris(dimethyl-amino)phosphoniumhexafluorophosphate, HF refers to hydrofluoric acid, TEA refers totriethylamine, TFA refers to trifluoroacetic acid, PCC refers topyridinium chlorochromate.

Generally, the compounds of formula (I) are prepared by reacting acompound of formula (II) with a compound of formula (III):

wherein R¹, R², R³, R⁴ and X are as defined in formula (I), with anyreactive functional groups protected, in the presence of EDC and HOBT;

and thereafter removing any protecting groups, and optionally forming apharmaceutically acceptable salt.

In particular, compounds of formula (I) are prepared by the generalmethods described in Scheme I.

Reagents and conditions: (a) PhSO₂Cl, pyridine, t-BuOH; (b)N-bromosuccinamide, benzoyl peroxide, CH₂Cl₂; (c) 40% CH₃NH₂ in H₂O; (d)(CH₃CO)₂O, (i-Pr)₂NEt, CH₂Cl₂; (e) H₂, 10% Pd/C, MeOH; (f) TFA, CH₂Cl₂;(g) 1-methyl-2-(methylaminomethyl)indole, EDC, HOBt.H₂O. (i-Pr)₂NEt,DMF.

Commercially available 3-methyl-4-nitrobenzoic acid (I-1) is protectedat the carboxylic acid functionality with a suitable protecting group,for instance a tertiary butyl (t-Bu) group, to afford I-2. The use ofprotecting groups to mask reactive functionality is well-known to thoseof skill in the art, and other protecting groups are listed in standardreference volumes, such as Greene, “Protective Groups in OrganicSynthesis” (published by Wiley-Interscience). The benzylic position isbrominated under radical conditions using N-bromosuccinamide (NBS) andthe radical initiator benzoyl peroxide affording I-3. Halogenation ofreactive positions, such as the benzylic position of I-2, is well known.Nucleophilic substitution of the bromide is accomplished with an excessof aqueous methyl amine, for instance 40% methylamine in water,providing the benzyl amine I-4. Acylation of the pendent nitrogen isaccomplished with a suitable acylating reagent such as an acyl halide,or an acid anhydride, to afford N-acyl-substituted derivatives. Forexample, the benzyl nitrogen can be acylated with acetic anhydride, toafford the amide derivative I-5. The aryl nitro compound I-5 isconverted under hydrogenation conditions, in the presence of a catalyticamount of palladium metal on activated carbon (Pd/C), to the amine I-6.Aromatic nitro groups can be reduced by a number of methods involvingthe use of metals and example procedures can be found in standardchemistry texts such as, “Reductions in Organic Chemistry” published bythe (American Chemical Society). The t-butyl ester is removed to revealthe carboxylic acid functionality with a suitable acid reagent such astrifluoroacetic acid (TFA) to give I-7. Other standard methods forremoval of a t-butyl protecting group are described by Greene (seeabove). The carboxylic acid derivative is then converted to amide I-8 byreaction with an activating agent and a suitable amine species. Forexample, acid I-7 is converted to an activated form by reaction with EDCand HOBt, and the activated form is subsequently reacted with amine[1-methyl-2-(methylaminomethyl)indole] in a suitable solvent such asDMF, CH₂Cl₂, or CH₃CN. Depending on whether acid neutralization isrequired, an added base, such as triethylamine (Et₃N),diisopropylethylamine ((i-Pr)₂NEt), or pyridine, may be used.

Many additional methods for converting a carboxylic acid to an amide areknown, and can be found in standard reference books, such as “Compendiumof Organic Synthetic Methods”. Vol. I-VI (published byWiley-Interscience), or Bodansky, “The Practice of Peptide Synthesis”(published by Springer-Verlag), which are incorporated herein byreference.

Amide coupling reagents as used herein denote reagents which may be usedto form peptide bonds. Typical coupling methods employ carbodiimides,activated anhydrides and esters and acyl halides. Reagents such as EDC,DCC, DPPA, PPA, BOP reagent, HOBt, N-hydroxysuccinimide and oxalylchloride are typical.

Typically, the amine is coupled via its free amino group to anappropriate carboxylic acid substrate using a suitable carbodiimidecoupling agent, such as N,N′dicyclohexyl carbodiimide (DCC), optionallyin the presence of catalysts such as 1-hydroxybenzotriazole (HOBt) anddimethylamino pyridine (DMAP). Other methods, such as the formation ofactivated esters, anhydrides or acid halides, of the free carboxyl of asuitably protected acid substrate, and subsequent reaction with the freeamine, optionally in the presence of a base, are also suitable. Forexample, a benzoic acid is treated in an anhydrous solvent, such asmethylene chloride or tetrahydrofuran (THF), in the presence of a base,such as N-methylmorpholine, DMAP or a trialkylamine, with isobutylchloroformate to form the “activated anhydride”, which is subsequentlyreacted with the free amine.

Acid addition salts of the compounds are prepared in a standard mannerin a suitable solvent from the parent compound and an excess of an acid,such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, phosphoric,acetic, trifluoroacetic, maleic, succinic or methanesulfonic. Certain ofthe compounds form inner salts or zwitterions which may be acceptable.Cationic salts are prepared by treating the parent compound with anexcess of an alkaline reagent, such as a hydroxide, carbonate oralkoxide, containing the appropriate cation; or with an appropriateorganic amine. Cations such as Li⁺, Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺ and NH₄ ⁺ arespecific examples of cations present in pharmaceutically acceptablesalts.

This invention also provides a pharmaceutical composition whichcomprises a compound according to formula (I) and a pharmaceuticallyacceptable carrier. Accordingly, the compounds of formula (I) may beused in the manufacture of a medicament. Pharmaceutical compositions ofthe compounds of formula (I) prepared as hereinbefore described may beformulated as solutions or lyophilized powders for parenteraladministration. Powders may be reconstituted by addition of a suitablediluent or other pharmaceutically acceptable carrier prior to use. Theliquid formulation may be a buffered, isotonic, aqueous solution.Examples of suitable diluents are normal isotonic saline solution,standard 5% dextrose in water or buffered sodium or ammonium acetatesolution. Such formulation is especially suitable for parenteraladministration, but may also be used for oral administration orcontained in a metered dose inhaler or nebulizer for insufflation. Itmay be desirable to add excipients such as polyvinylpyrrolidone,gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol,sodium chloride or sodium citrate.

Alternately, these compounds may be encapsulated, tableted or preparedin a emulsion or syrup for oral administration. Pharmaceuticallyacceptable solid or liquid carriers may be added to enhance or stabilizethe composition, or to facilitate preparation of the composition. Solidcarriers include starch, lactose, calcium sulfate dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. Liquid carriers include syrup, peanut oil, olive oil, salineand water. The carrier may also include a sustained release materialsuch as glyceryl monostearate or glyceryl distearate, alone or with awax. The amount of solid carrier varies but, preferably, will be betweenabout 20 mg to about 1 g per dosage unit. The pharmaceuticalpreparations are made following the conventional techniques of pharmacyinvolving milling, mixing, granulating, and compressing, when necessary,for tablet forms; or milling, mixing and filling for hard gelatincapsule forms. When a liquid carrier is used, the preparation will be inthe form of a syrup, elixir, emulsion or an aqueous or non-aqueoussuspension. Such a liquid formulation may be administered directly p.o.or filled into a soft gelatin capsule.

For rectal administration, the compounds of this invention may also becombined with excipients, such as cocoa butter, glycerin, gelatin orpolyethylene glycols, and molded into a suppository.

For topical administration, the compounds of this invention may becombined with diluents to take the form of ointments, gels, pastes,creams, powders or sprays. The compositions which are ointments, gels,pastes or creams contain diluents, for example animal and vegetablefats, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures of these substances. The compositions which arepowders or sprays contain diluents, for example, lactose, talc, silicicacid, aluminum hydroxide, calcium silicate and polyamide powder, ormixtures of these substances. Additionally, for topical ophthalmologicadministration, the typical carriers are water, mixtures of water andwater miscible solvents, such as lower alkanols or vegetable oils, andwater-soluble non-toxic polymers, for example cellulose derivatives,such as methyl cellulose.

The compounds described herein are inhibitors of FabI, and are usefulfor treating bacterial infections. For instance, these compounds areuseful for the treatment of bacterial infections, such as, for example,infections of upper respiratory tract (e.g. otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.empyema, lung abscess), cardiac (e.g. infective endocarditis),gastrointestinal (e.g. secretory diarrhoea, splenic abscess,retroperitoneal abscess), CNS (e.g. cerebral abscess), eye (e.g.blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.epididymitis, intrarenal and perinephric abscess, toxic shock syndrome),skin (e.g. impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis), and bone and joint (e.g. septicarthritis, osteomyelitis). Also, the compounds of this invention may beuseful as antifungal agents. Additionally, the compounds may be usefulin combination with known antibiotics.

The compounds of this invention are administered to the patient, in amanner such that the concentration of drug is sufficient to treatbacterial infections. The pharmaceutical composition containing thecompound is administered at an oral dose of between about 10 mg to about1000 mg, taken once or several times daily, in a manner consistent withthe condition of the patient. Preferably, the oral dose would be about50 mg to about 500 mg, although the dose may be varied depending uponthe age, body weight and symptoms of the patient. For acute therapy,parenteral administration is preferred. An intravenous infusion of thecompound of formula (I) in 5% dextrose in water or normal saline, or asimilar formulation with suitable excipients, is most effective,although an intramuscular bolus injection is also useful. The preciselevel and method by which the compounds are administered is readilydetermined by one skilled in the art.

The compounds may be tested in one of several biological assays todetermine the concentration of compound which is required to have agiven pharmacological effect.

Cloning of S. aureus FabI:

The fabI gene was cloned from the chromosomal DNA of S. aureus strainWCUH29 using the polymerase chain reaction. Amplification was performedusing Taq DNA polymerase (BRL) and the following primers:5′-CGCCTCGAGATGTTAAATCTTGAAAACAAAACATATGTC-3′ and5′-CGCGGATCCAATCAAGTCAGGTTGAAATATCCA-3′ (XhoI and BamHI sitesunderlined). The resulting fragment was then digested with XhoI andBamHI and ligated into XhoI- and BamHI-digested expression vectorpET-16b (Novagen), producing pET-His₁₀-fabI. The gene sequence of fabIwas confirmed by automated cycle sequencing using an Applied Biosystemsmodel 377 machine. The untagged version of pET-fabI was constructed bydigesting pET-His₁₀-fabI with NcoI and NdeI to remove a 97 bp fragmentencoding the His 10 tag, the factor Xa cleavage site and the first 8amino acids of FabI, and replacing it with a linker encoding the first 8amino acids of FabI plus a glycine residue between the initiatormethionine and the lysine at position 2. This plasmid was calledpET-fabI. The linker was made by annealing the following twooligonucleotides: 5′-CATGGGCTTAAATCTTGAAAACAAAACA-3′ and5′-TATGTTTTGTTTTCATTTAAGCC-3′. The linker sequence in pET-fabI wasconfirmed by dideoxy sequencing. Only native FabI was used for compoundevaluation. For overproduction of native FabI, plasmid pET-fabI wastransformed into BL21(DE3) (Novagen) cells, to form strainBL21(DE3):pET-fabI.

Purification of S. aureus FabI

S. aureus FabI was expressed as soluble protein to 10% of total cellprotein, 400 g cells being recovered from 15 L fermentation in tryptonephosphate medium. The cells were lysed and the sample centrifuged. Theresulting supernatant was filtered and purified using three consecutivechromatography columns: ion-exchange (Sourse 15Q), dye-affinity (Bluesepharose), and size exclusion chromatography columns (Superose 12).After each column the FabI containing fractions were pooled,concentrated, and checked for purity and biological activity.

Cloning of E. coli FabI:

A PCR fragment of correct size for E. coli FabI was PCR amplified fromE. coli chromosomal DNA. subcloned into the TOPO TA cloning vector, andverified by colony PCR+ restriction endonuclease analysis. Thepresumptive E. coli FabI PCR fragment was subcloned into the expressionvector pBluePet. The FabI clone was transformed into E. coli strainBL21(DE3). Small Scale expression studies show an over-expressed proteinband of correct molecular weight (˜28 Kda) for E. coli FabI clearlyvisible following Coomassie staining of SDS PAGE gels. DNA sequencing ofthe E. coli FabI expression constructs illustrated that no errors wereapparent. N′ terminal amino acid sequencing has confirmed theover-expressed protein band to be E. coli FabI.

Purification of E. coli FabI

E. coli FabI was expressed as soluble protein to 15% of total cellprotein, 120 g cells being recovered from 3 L fermentation in shakeflasks in modified terrific broth. The cells were lysed and the samplecentrifuged. The resulting supernatant was filtered and purified usingthree consecutive chromatography columns: ion-exchange (Sourse 15Q),dye-affinity (blue sepharose), and size exclusion (superose 12). Aftereach column the FabI containing fractions were pooled, concentrated andchecked for purity and biological activity.

S. aureus FabI Enzyme Inhibition Assay (NADH):

Assays were carried out in half-area, 96-well microtitre plates.Compounds were evaluated in 50-uL assay mixtures containing 100 mMNaADA, pH 6.5 (ADA=N-[2-acetamido]-2-iminodiacetic acid), 4% glycerol,0.25 mM crotonoyl CoA, 1 mM NADH, and an appropriate dilution of S.aureus FabI. Inhibitors were typically varied over the range of 0.01-10uM. The consumption of NADH was monitored for 20 minutes at 30° C. byfollowing the change in absorbance at 340 nm. Initial velocities wereestimated from an exponential fit of the non-linear progress curvesrepresented by the slope of the tangent at t=0 min. IC₅₀'s wereestimated from a fit of the initial velocities to a standard,4-parameter model and are typically reported as the mean±S.D. ofduplicate determinations. Triclosan, a commercial antibacterial agentand inhibitor of FabI, is currently included in all assays as a positivecontrol. Compounds of this invention have IC₅₀'s from about 2.0micromolar to about 0.15 micromolar.

S. aureus FabI Enzyme Inhibition Assay (NADPH):

Assays were carried out in half-area, 96-well microtitre plates.Compounds were evaluated in 150-uL assay mixtures containing 100 mMNaADA, pH 6.5 (ADA=N-[2-acetamido]-2-iminodiacetic acid), 4% glycerol,0.25 mM crotonoyl CoA, 50 uM NADPH, and an appropriate dilution of S.aureus FabI. Inhibitors were typically varied over the range of 0.01-10uM. The consumption of NADPH was monitored for 20 minutes at 30° C. byfollowing the change in absorbance at 340 nm. Initial velocities wereestimated from an exponential fit of the non-linear progress curvesrepresented by the slope of the tangent at t=0 min. IC₅₀'s wereestimated from a fit of the initial velocities to a standard,4-parameter model and are typically reported as the mean±S.D. ofduplicate determinations. Triclosan, a commercial antibacterial agentand inhibitor of FabI, is currently included in all assays as a positivecontrol.

E. coli FabI Enzyme Inhibition Assay:

Assays were carried out in half-area, 96-well microtitre plates.Compounds were evaluated in 150-uL assay mixtures containing 100 mMNaADA, pH 6.5 (ADA=N-[2-acetamido]-2-iminodiacetic acid), 4% glycerol,0.25 mM crotonoyl CoA, 50 uM NADH, and an appropriate dilution of E.coli FabI. Inhibitors were typically varied over the range of 0.01-10uM. The consumption of NADH was monitored for 20 minutes at 30° C. byfollowing the change in absorbance at 340 nm. Initial velocities wereestimated from an exponential fit of the non-linear progress curvesrepresented by the slope of the tangent at t=0 min. IC₅₀'s wereestimated from a fit of the initial velocities to a standard,4-parameter model and are typically reported as the mean±S.D. ofduplicate determinations. Triclosan, a commercial antibacterial agentand inhibitor of FabI, is currently included in all assays as a positivecontrol. Compounds of this invention have IC₅₀'s from about 4.0micromolar to about 0.15 micromolar.

Preparation and Purification of Crotonoyl-ACP:

Reactions contained 5 mg/mL E. coli apo-ACP, 0.8 mM crotonoyl-CoA(Fluka), 10 mM MgCl₂, and 30 uM S. pneumoniae ACP synthase in 50 mMNaHEPES, pH 7.5. The mixture was gently mixed on a magnetic stirrer at23° C. for 2 hr, and the reaction was terminated by the addition of 15mM EDTA. The reaction mixture was filtered through a 0.2 micron filter(Millipore) and applied to a MonoQ column (Pharmacia) equilibrated with20 mM Tris-Cl, pH 7.5. The column was washed with buffer until allnon-adherent material was removed (as observed by UV detection), and thecrotonoyl-ACP was eluted with a linear gradient of 0 to 400 mM NaCl.

S. aureus FabI Enzyme Inhibition Assay Using Crotonoyl-ACP:

Assays are carried out in half-area, 96-well microtitre plates.Compounds are evaluated in 150 uL assay mixtures containing 100 mMNaADA, pH 6.5 (ADA=N-(2-acetamido)-2-iminodiacetic acid), 4% glycerol,25 uM crotonoyl-ACP, 50 uM NADPH, and an appropriate dilution of S.aureus FabI (approximately 20 nM). Inhibitors are typically varied overthe range of 0.01-10 uM. The consumption of NADPH is monitored for 20minutes at 30° C. by following the change in absorbance at 340 nm.Initial velocities are estimated from a linear fit of the progresscurves. IC50's are estimated from a fit of the initial velocities to astandard, 4-parameter model (Equation 1) and are typically reported asthe mean±S.D. of duplicate determinations. The apparent Ki is calculatedfrom Equation assuming the inhibition is competitive with crotonoyl-ACP.

v=Range/(1+[I]/IC50)s+Background  Equation 1:

Ki(app)=IC50/(1+[S]/Ks)  Equation 2:

Antimicrobial Activity Assay:

Whole-cell antimicrobial activity was determined by broth microdilutionusing the National Committee for Clinical Laboratory Standards (NCCLS)recommended procedure, Document M7-A4, “Methods for DilutionSusceptibility Tests for Bacteria that Grow Aerobically”. The compoundwas tested in serial two-fold dilutions ranging from 0.06 to 64 mcg/mL.Test organisms were selected from the following laboratory strains:

Staphylococcus aureus Oxford Staphylococcus aureus WCUH29, Streptococcuspneumoniae ERY2, Streptococcus pneumoniae 1629, Streptococcus pneumoniaeN 1387, Enterococcus faecalis 1, Enterococcus faecalis 7, Haemophilusinfluenzae Q1, Haemophilus influenzae NEMCI, Moraxella Catarrhalis 1502,Escherichia coli 7623 AcrABEFD+, Escherichia coli 120 AcrAB−,Escherichia coli MG1655, Escherichia coli MG1658. The minimum inhibitoryconcentration (MIC) was determined as the lowest concentration ofcompound that inhibited visible growth. A mirror reader was used toassist in determining the MIC endpoint.

One skilled in the art would consider any compound with a MIC of lessthan 256 μg/mL to be a potential lead compound. Preferably, thecompounds used in the antimicrobial assays of the present invention havea MIC value of less than 128 μg/mL. Most preferably, said compounds havea MIC value of less than 64 μg/mL.

The examples which follow are intended in no way to limit the scope ofthis invention, but are provided to illustrate how to make and use thecompounds of this invention. Many other embodiments will be readilyapparent to those skilled in the art.

EXAMPLES General

Proton nuclear magnetic resonance (¹H NMR) spectra were recorded at 300MHz, and chemical shifts are reported in parts per million (δ) downfieldfrom the internal standard tetramethylsilane (TMS). Abbreviations forNMR data are as follows: s=singlet, d=doublet, t=triplet, q=quartet,m=multiplet, dd=doublet of doublets, dt=doublet of triplets,app=apparent, br=broad. J indicates the NMR coupling constant measuredin Hertz. CDCl₃ is deuteriochloroform, DMSO-d₆ ishexadeuteriodimethylsulfoxide, and CD₃OD is tetradeuteriomethanol. Massspectra were obtained using electrospray (ES) ionization techniques.Elemental analyses were performed by Quantitative Technologies Inc.,Whitehouse, N.J. Melting points were obtained on a Thomas-Hoover meltingpoint apparatus and are uncorrected. All temperatures are reported indegrees Celsius. Analtech Silica Gel GF and E. Merck Silica Gel 60 F-254thin layer plates were used for thin layer chromatography. Flashchromatography was carried out on E. Merck Kieselgel 60 (230-400 mesh)silica gel. Analytical HPLC was performed on Beckman chromatographysystems. Preparative HPLC was performed using Gilson chromatographysystems. ODS refers to an octadecylsilyl derivatized silica gelchromatographic support. YMC ODS-AQ® is an ODS chromatographic supportand is a registered trademark of YMC Co. Ltd., Kyoto, Japan. PRP-1® is apolymeric (styrene-divinylbenzene) chromatographic support, and is aregistered trademark of Hamilton Co., Reno, Nev. Celite® is a filter aidcomposed of acid-washed diatomaceous silica, and is a registeredtrademark of Manville Corp., Denver, Colo.

Preparation 1

Preparation of 1-methyl-2-(methylaminomethyl)indole

a) Ethyl 1-methylindole-2-carboxylate

NaH (60% dispersion in mineral oil, 8.0 g, 200.5 mmole) was washed withhexanes, then was suspended in dry DMF (530 mL). Solid ethylindole-2-carboxylate (25.3 g, 133.7 mmole) was added portionwise over5-10 min, allowing gas evolution to subside between additions. When theaddition was complete, the yellow mixture was stirred for 15 min, thenmethyl iodide (42 mL, 668.3 mmole) was added all at once. The reactionwas exothermic, and the internal temperature rose to 40-45° C. After 1hr, the reaction was quenched with 10% NH₄Cl (100 mL) and concentratedon the rotavap (high vacuum). The residue was partitioned between Et₂O(500 mL) and H₂O (100 mL), and the layers were separated. The Et₂O layerwas washed with H₂O (100 mL). dried (MgSO₄), and concentrated to leavethe title compound (27.1 g, quantitative) as a light yellow solid. Thiswas used without further purification: TLC (10% EtOAc/hexanes)R_(f)=0.39.

b) N,1-Dimethylindole-2-carboxamide

A suspension of ethyl 1-methylindole-2-carboxylate (27.1 g, 133.3 mmole)in 40% aqueous CH₃NH₂ (300 mL) and MeOH (30 mL) was stirred at RT. Asolid tended to gradually creep up the walls of the flask, and waswashed down periodically with MeOH. The flask was tightly stoppered tokeep the material inside the flask. As the reaction proceeded, the soliddissolved, but eventually the product began to precipitate. The reactionwas stirred at RT for 3 days, then was concentrated to removeapproximately 200 mL of the solvent. The remaining residue was dilutedwith H₂O (300 mL), and the solid was collected by suction filtration andwashed with H₂O. Drying at 50-60° C. in high vacuum left the titlecompound (23.4 g, 93%) as a faintly yellow solid: MS (ES) m/e 189(M+H)⁺.

c) 1-Methyl-2-(methylaminomethyl)indole

A 3-liter 3-necked roundbottom flask equipped with overhead stirring wascharged with N,1-dimethylindole-2-carboxamide (23.4 g, 124.6 mmole) andanhydrous THF (170 mL). The solution was stirred while a solution ofLiAlH₄ in THF (1.0 M, 250 mL, 250 mmole) was added via syringe. Gas wasevolved during the addition of the first 50 mL of LiAlH₄ solution. Whenthe addition was complete, the resulting light yellow solution washeated at gentle reflux. After 24 hr, the reaction was cooled in ice andquenched by the sequential dropwise addition of H₂O (9.5 mL), 15% NaOH(9.5 mL), and H₂O (28.5 mL).

The mixture was stirred for 15 min, then was filtered through celite®,and the filter pad was washed thoroughly with THF. The filtrate wasconcentrated and the residue was flash chromatographed on silica gel(10% MeOH/CHCl₃ containing 5% conc. NH₄OH). The title compound (20.2 g,93%) was obtained as a light yellow oil: MS (EI) m/e 175 (M+H)⁺.

Preparation 2

The Preparation of 1-Methyl-3-(methylaminomethyl)indole

a) Ethyl 1-methylindole-3-carboxylate

According to the procedure of Preparation 1a, except substituting ethyl3-indole acetate (25.3 g, 133.7 mmole) for ethyl indole-2-carboxylate,the title compound was prepared as a light yellow solid and used withoutfurther purification.

b) N,1-Dimethylindole-3-carboxamide

According to the procedure of Preparation 1b, except substituting ethyl1-methylindole-3-carboxylate (27.1 g, 133.3 mmole) for ethyl1-methylindole-2-carboxylate, the title compound (23.4 g, 93%) wasprepared as a light yellow solid and used without further purification:MS (ES) m/e 189 (M+H)⁺.

c) 1-Methyl-3-(methylaminomethyl)indole

According to the procedure of Preparation 1c, except substitutingN,1-dimethylindole-3-carboxamide (23.4 g, 124.6 mmole) forN,1-dimethylindole-2-carboxamide, the title compound (20.2 g, 93%) wasprepared as a light yellow oil: MS (ES) m/e 175 (M+H)⁺.

Preparation 3

Preparation of 4-amino-3-[(N-methylacetylamino)methyl]benzoic acidtrifluoroacetate

a) tert-Butyl 3-methyl-4-nitro benzoate

To a pyridine solution of 3-methyl-4-nitrobenzoic acid (18.1 g, 100.0mmole) at RT was added benzenesulfonyl chloride in one portion. After 10min. anhydrous t-butanol (9.4 mL, 100.0 mmole) was added and thereaction solution was allowed to stir for 1 hr. The resulting suspensionwas poured into ice-water (400 mL) and stirred vigorously for 1 hr. Thelight yellow suspension was filtered through a scinter-glass funnelwashing with H₂O. The remaining yellow solids were dissolved in toluene(400 mL), dried over MgSO₄ and then filtered through a short column ofsilica gel washing with toluene. Concentration of the solution undervacuum (15 mm Hg) and drying on high vacuum afforded the tide compound(22.5 g, 95%) as a light yellow solid: MS (ES) m/e 238 (M+H)⁺.

b) tert-Butyl 3-methylaminomethyl-4-nitro benzoate

To a stirred solution of tert-butyl 3-methyl-4-nitro benzoate (22.5 g,94.9 mmole) in CH₂Cl₂ (450 mL) at RT contained in a 1 L one-neck roundbottom flask was added NBS (18.6 g, 104.4 mmole) and benzoyl peroxide(2.3 g, 9.5 mmole). The flask was equipped with a reflux condenser and a150 watt tungsten-light source was shined on the reaction for 36 hrs.The reaction solution was washed with H₂O, dried over Na₂SO₄ andconcentrated under vacuum. The resulting orange residue was dissolved inethyl acetate and filtered through silica gel (EtOAc/hexanes, 1:4).Concentration on a rotary evaporator gave an orange oil which was useddirectly in the next step without further purification.

To the crude bromide in THF (150 mL) was added 40% aqueous CH₃NH₂ (50mL) in one portion at RT. After 18 hr, the reaction solution wasconcentrated to remove the THF. The resulting aqueous solution wasextracted with EtOAc (2×200 mL) and the combined organic phases weresequentially washed with H₂O and brine. Drying over K₂CO₃ andconcentration gave a yellow oil which was purified on silica(hexanes/EtOAc, 1:1) to afford the title compound (19.2 g, 76%) as alight yellow solid: MS (ES) m/e 267 (M+H)⁺.

c) tert-Butyl-4-amino-3-[(N-methylacetylamino)methyl]benzoate

To a stirred solution of t-butyl 3-(methylaminomethyl)-4-nitro benzoate(2.4 g, 9.0 mmole) in CH₂Cl₂ at RT was added Et₃N (2.52 mL, 18.0 mmole)and acetic anhydride (1.8 g, 18.0 mmole). After 12 hrs. the reactionsolution was concentrated under vacuum, dissolved in EtOAc (150 mL) andwashed with H₂O. The EtOAc solution was dried over Na₂SO₄ andconcentrated under vacuum to a yellow oil. The oil was further driedunder high vacuum and used directly in the next procedure.

The crude product was dissolved in EtOAc (50 mL) and CH₃OH (50 mL) andplaced in a Parr hydrogenation flask. A catalytic amount of 10% Pd/C wasadded to the reaction solution and the contents were placed on a Parrshaker under H₂ (50 psi) and shaken for 4 hours. The solution wasfiltered through celite and concentrated under vacuum. Purification onsilica (hexanes/EtOAc, 1:1) afforded the title compound (2.13 g, 85%over two steps) as an off-white solid: MS (ES) m/e 279 (M+H)⁺.

d) 4-amino-3-[(N-methylacetylamino)methyl]benzoic acid trifluoroacetatesalt

To a solution of t-butyl 4-amino-3-[(N-methylacetylamino)methyl]benzoate(2.13 g, 7.65 mmole) in CH₂Cl₂ (150 mL) at RT was added TFA (30 mL).After 12 hr, the reaction solution was concentrated to an oil and driedunder high vacuum overnight. The resulting residue was washed withhexanes and Et₂O to afford the title compound (2.56 g, 7.60 mmole) as anoff-white solid. This compound was used without further purification: MS(ES) m/e 223 (M+H-TFA)⁺.

Preparation 4

Preparation of 4-amino-3-[(N-phenethylacetylamino)methyl]benzoic acid

a) tert-Butyl 4-nitro-3-(phenethylamino)methyl benzoate

Crude tert-butyl 3-bromomethyl-4-nitrobenzoate (from Preparation 3b) wasdissolved in dry THF (50 mL), and solid NaHCO₃ (2.52 g, 30 mmole) wasadded. The mixture was stirred briskly, and phenethylamine (3.8 mL, 30mmole) was added. The color of the solution darkened slightly to adeeper yellow. Within several minutes, the mixture had become verycloudy. After 4 hr, the reaction was concentrated and the residue waspartitioned between H₂O (50 mL) and Et₂O (100 mL). The layers wereseparated, and the aqueous layer was extracted with Et₂O (2×100 mL). Theorganic layers were combined, dried (MgSO₄), and concentrated to ayellow oil. Flash chromatography on silica gel (20% EtOAc/hexanes) gavethe title compound (2.86 g, 40% for two steps) as a yellow oil: ¹H NMR(250 MHz, CDCl₃) δ 8.19 (d, J=1.7 Hz, 1H), 7.98 (dd, J=8.4, 1.7 Hz, 1H),7.90 (d. J=8.4 Hz, 1H), 7.10-7.40 (m, 5H), 4.06 (s, 2H), 2.75-3.00 (m,4H), 1.61 (s, 9H); MS (ES) m/e 357 (M+H)⁺.

b) tert-Butyl3-[N-(tert-butoxycarbonyl)-N-phenethylamino]methyl-4-nitrobenzoate

Di-tert-butyl dicarbonate (2.10 g, 9.62 mmole) was added all at once toa solution of tert-butyl 3-(phenethylamino)methyl-4-nitrobenzoate (2.86g, 8.02 mmole) in CHCl₃ (30 mL) at RT. The reaction was stirred at RTFor 2.5 hr, then at reflux for 0.5 hr. Concentration and flashchromatography on silica gel (15% EtOAc/hexanes) gave the title compound(3.70 g, quantitative) as a yellow oil: ¹H NMR (250 MHz, CDCl₃) δ7.85-8.10 (m, 3H), 7.05-7.40 (m, 5H), 4.55-4.85 (m, 2H), 3.35-3.60 (m,2H), 2.75-3.00 (m, 2H), 1.20-1.80 (m, 18H); MS (ES) m/e 479 (M+Na)⁺, 457(M+H)⁺.

c) tert-Butyl4-amino-3-[[N-(tert-butoxycarbonyl)-N-phenethylamino]methyl]benzoate

A mixture of tert-butyl3-[[N-(tert-butoxycarbonyl)-N-phenethylamino]methyl]-4-nitrobenzoate(2.7 g, 5.9 mmole), 10% Pd/C (0.6 g, 0.6 mmole Pd), and EtOAc (60 mL)was shaken under H₂ (50 psi). After 3 hr, the mixture was filtered toremove the catalyst, and the filtrate was concentrated to dryness. Flashchromatography on silica gel (20% EtOAc/hexanes) gave the title compound(2.3 g, 91%) as a yellow foamy oil which slowly partially solidified: ¹HNMR (250 MHz, CDCl₃) δ 7.74 (dd, J=8.4, 2.0 Hz, 1H), 7.67 (d, J=2.0 Hz,1H), 7.05-7.40 (m, 5H), 6.57 (d, J=8.4 Hz, 1H), 5.00 (br s, 2H), 4.30(s, 2H), 3.32 (app. t, 2H), 2.69 (app. t, 2H), 1.59 (s, 9H), 1.46 (s,9H); MS (ES) m/e 449.2 (M+Na)⁺, 427.2 (M+H)⁺.

d) 4-amino-3-[N-phenethylaminomethyl]benzoate trifluoroacetate salt

To a solution of tert-butyl4-amino-3-[[N-(tert-butoxycarbonyl)-N-phenethylamino]methyl]benzoate(2.3 g, 5.4 mmole) in CH₂Cl₂ (100 mL) at RT was added TFA (25 mL). After12 hr, the reaction solution was concentrated to an oil and the residuedried under high vacuum overnight. The resulting residue was washed withhexanes and diethyl ether to afford the title compound (2.7 g, 5.4mmole) as an off-white solid: MS (ES) m/e 271 (M+H-2TFA)⁺.

e) 4-amino-3-[(N-phenethylacetylamino)methyl]benzoic acid

To a stirred solution of 4-amino-3-[N-phenethylaminomethyl]benzoatebis-trifluoroacetate salt (1.0 g, 2.0 mmole) in CH₂Cl₂ at RT was addedEt₃N (1.1 mL, 7.9 mmole) and acetic anhydride (0.26 g, 2.6 mmole). After12 hr, the reaction solution was concentrated under vacuum, dissolved in1M NaOH (15 mL), and washed with hexanes. The aqueous solution wasneutralized with 1M HCl and extracted with EtOAc (2×50 mL). The EtOAcsolution was dried over Na₂SO₄ and concentrated to an off-white solidwhich was used without further purification: MS (ES) m/e 313 (M+H)⁺.

Preparation 5

Preparation of tert-Butyl-4-amino-3-[(N-methyl)aminomethyl)]benzoate

A solution of t-butyl-3-[N-(methyl)aminomethyl]-4-nitro-benzoate (12.0g, 45.1 mmole), from procedure 3a, in CH₃OH (50 mL) was placed in a Parrhydrogenation flask. Approximately (0.50 g) of 10% Pd/C was added to thereaction solution and the contents were shaken on a Parr shaker under H₂(50 psi) for 4 hours. The suspension was filtered through celite andconcentrated under vacuum. Washing of the resulting residue with Et₂Oafforded a viscous light yellow oil which was used without furtherpurification: MS (ES) m/e 237 (M+H)⁺.

Preparation 6

Preparation of4-amino-3-[(2-hydroxy-4,N-dimethylpentanoylamino)methyl]benzoic acidtrifluoroacetate

a)tert-Butyl-4-amino-3-[(2-hydroxy-4,N-dimethylpentanoylamino)methyl]benzoicacid

To a stirred solution of t-butyl-3-[N-(methyl)aminomethyl]-4-aminobenzoate (2.6 g, 11.0 mmole) in DMF (20 mL) at RT was addeddiisopropylethyl amine (2.9 mL, 16.9 mmole), HOBt (2.3 g, 16.9 mmole)2-hydroxy-4-methylpentanoic acid (2.04 g, 15.15 mmole) and finally EDC(3.24 g, 16.9 mmole). After 12 hr, the reaction contents were pouredonto H₂O (200 mL) and extracted with EtOAc (2×150 mL). The organicphases were combined and sequentially washed with H₂O (100 mL) andbrine. Drying over Na₂SO₄ and purification on silica (CHCl₃/CH₃OH, 95:5)afforded the title compound (3.50 g, 91%) as a light yellow oil: MS (ES)m/e 351 (M+H)⁺.

b) 4-Amino-3-[(2-hydroxy-4,N-dimethylpentanoylamino)methyl]benzoic acidtrifluoroacetate

To a stirred solution oft-butyl-4-amino-3-[(2-hydroxy-4,N-dimethylpentanoylamino)methyl]benzoicacid (3.50 g, 10.0 mmole) in CH₂Cl₂ (20 mL) at RT was added TFA (20 mL).After 12 hr, the reaction contents were evaporated, washed with hexanesand dried under high vacuum overnight affording the title compound as anorange oil (4.42 g, 10.8 mmol). This product was used without furtherpurification: MS (ES) m/de 295 (M+H-TFA)⁺.

Preparation 7

Preparation of 4-amino-3-[(ethoxy-N-methylcarbonylamino)methyl]benzoicacid trifluoro acetate

a) tert-Butyl 4-amino-3-[(ethoxy-N-methylcarbonylamino)methyl]benzoate

To a stirred solution of t-butyl 3-[N-(methyl)aminomethyl]-4-aminobenzoate (251 g, 10.63 mmole) in CH₂Cl₂ (20 mL) at 0° C. was addedtriethyl amine (1.63 mL, 11.7 mmole), and ethylchloroformate (1.15 g,10.63 mmole). The reaction solution was allowed to warm to RT overnight.The reaction contents were concentrated under vacuum and partitionbetween H₂O (100 mL) and EtOAc (200 mL). The organic phase was combinedwashed sequentially with H₂O (100 mL) and brine. Drying over Na₂SO₄ andpurification on silica (CHCl₃/CH₃OH, 95:5) afforded the title compound(2.98 g, 91%) as a light yellow oil: MS (ES) m/e 309 (M+H)⁺.

b) 4-amino-3-[(ethoxy-N-methylcarbonylamino)methyl]benzoic acidtrifluoro acetate

To a stirred solution oft-butyl-4-amino-3-[(2-hydroxy-4,N-dimethylpentanoylamino)methyl]benzoicacid (2.98 g, 9.67 mmole) in CH₂Cl₂ (20 mL) at RT was added TFA (20 mL).After 12 hr, the reaction contents were concentrated, washed withhexanes and dried under high vacuum overnight affording the titlecompound as an orange oil (3.51 g, 9.60 mmol). This product was usedwithout further purification: MS (ES) m/e 253 (M+H-TFA)⁺.

Preparation 8

Preparation of 4-amino-3-[(2-hydroxy-N-methylacetylamino)methyl]benzoicacid

a) tert-Butyl 4-nitro-3-[(2-hydroxy-N-methylacetylamino)methyl]benzoate

To a stirred solution of t-butyl-3-[N-(methyl)aminomethyl]-4-aminobenzoate (7.0 g, 26.3 mmole) in DMF (30 mL) at RT was addeddiisopropylethyl amine (5.0 mL, 28.9 mmole), HOBt (3.90 g, 28.9 mmole),2-hydroxyacetic acid (2.2 g, 28.9 mmole) and finally EDC (5.54 g, 28.9mmole). After 12 hr, the reaction contents were poured onto H₂O (200 mL)and extracted with EtOAc (2×150 mL). The organic phases were combinedand sequentially washed with H₂O (100 mL) and brine. Drying over Na₂SO₄and purification on silica (CHCl₃/CH₃OH, 95:5) afforded the titlecompound (7.92 g, 93%) as a light yellow oil: MS (ES) m/e 325 (M+H)⁺.

b) tert-Butyl 4-amino-3-[(2-hydroxy-N-methylacetylamino)methyl]benzoate

To a solution oft-butyl-4-nitro-3-[(2-hydroxy-N-methylacetylamino)methyl]benzoate (7.92g, 23.15 mmole) in CH₃OH (25 mL) and EtOAc (25 mL) in a Parrhydrogenation flask was added 10% Pd/C (0.20 g). The contents wereshaken on a Parr shaker under H₂ (50 psi) for 4 hours. The suspensionwas filtered through celite, concentrated under vacuum and washed withEt₂O to afford the title compound as a light orange foam which was usedwithout further purification: MS (ES) m/e 295 (M+H)⁺.

Preparation 9

Preparation of4-amino-3-[(2-hydroxy-3-indol-3-yl-N-methylpropanoylamino)methyl]benzoicacid trifluoro acetate

a) tert-Butyl4-amino-3-[(2-hydroxy-3-indol-3-yl-N-methylpropanoylamino)methyl]benzoicacid

To a stirred solution of t-butyl-3-[N-(methyl)aminomethyl]-4-aminobenzoate (0.60 g, 2.52 mmole), from Procedure 5, in DMF (20 mL) at RTwas added diisopropylethyl amine (0.88 mL, 5.03 mmole), HOBt (0.37 g,2.77 mmole), DL-3-indolelactic acid (0.57 g, 2.77 mmole) and finally EDC(0.53 g, 2.77 mmole). After 12 hr, the reaction contents were pouredonto H₂O (100 mL) and extracted with EtOAc (2×100 mL). The organicphases were combined and sequentially washed with H₂O (100 mL) andbrine. Drying over Na₂SO₄ and purification on silica (CHCl₃/CH₃OH, 95:5)afforded the title compound (0.99 g, 93%) as a light yellow oil: MS (ES)m/e 425 (M+H)⁺.

b)4-Amino-3-[(2-hydroxy-3-indol-3-yl-N-methylpropanoylamino)methyl]benzoicacid trifluoro acetate

To a stirred solution oft-butyl-4-amino-3-[(2-hydroxy-3-indol-3-yl-N-methylpropanoylamino)methyl]benzoicacid (0.99 g, 2.34 mmole) in CH₂Cl₂ (20 mL) at RT was added TFA (20 mL).After 12 hr, the reaction contents were evaporated, washed with Et₂O anddried under high vacuum overnight affording the title compound (0.86 g,2.34 mmol) as a pink solid. This product was used without furtherpurification: MS (ES) m/e 369 (M+H-TFA)⁺.

Preparation 10

Preparation of4-amino-3-[(2-cyclopentyl-N-methylacetylamino)methyl]benzoic acidtrifluoro acetate

a) tert-Butyl4-amino-3-[(2-cyclopentyl-N-methylacetylamino)methyl]benzoic acid

To a stirred solution of t-butyl-3-[N-(methyl)aminomethyl]-4-aminobenzoate (0.55 g, 2.32 mmole), from Procedure 5, in DMF (20 mL) at RTwas added diisopropylethyl amine (0.81 mL, 4.64 mmole), HOBt (0.34 g,2.55 mmole), cyclopentane acetic acid (0.33 g, 2.55 mmole) and finallyEDC (0.49 g, 2.55 mmole). After 12 hr, the reaction contents were pouredonto H₂O (100 mL) and extracted with EtOAc (2×100 mL). The organicphases were combined and sequentially washed with H₂O (100 mL) andbrine. Drying over Na₂SO₄ and purification on silica (CHCl₃/CH₃OH, 95:5)afforded the title compound (0.75 g, 94%) as a light yellow oil: MS (ES)m/e 348 (M+H)⁺.

b) 4-Amino-3-[(2-cyclopentyl-N-methylacetylamino)methyl]benzoic acidtrifluoro acetate

To a stirred solution oft-butyl-4-amino-3-[(2-cyclopentyl-N-methylacetylamino)methyl]benzoicacid (0.75 g, 2.16 mmole) in CH₂Cl₂ (20 mL) at RT was added TFA (20 mL).After 12 hr, the reaction contents were evaporated, washed with hexanesand dried under high vacuum overnight affording the title compound (0.63g, 2.16 mmol) as an orange oil. This product was used without furtherpurification: MS (ES) m/e 292 (M+H-TFA)⁺.

Preparation 11

Preparation of{4-amino-3-[(methylamino)methyl]phenyl}-N-methyl-N-[(1-methylindol-2-yl)methyl]carboxamide

a)t-Butyl-4-nitro-3-{[(N-methyl(phenylmethoxy)carbonylamino]methyl}benzoate

To a stirred solution of t-butyl-3-[N-(methyl)aminomethyl]-4-nitrobenzoate (11.97 g, 45.0 mmole), from Procedure 3b, in DMF (100 mL) at RTwas added triethyl amine (7.27 mL, 52.2 mmole), andN-(benzyloxycarbonyloxy)succinamide (13.0 g, 52.2 mmole). After 12 hr,the reaction contents were poured onto H₂O (200 mL) and extracted withEtOAc (2×200 mL). The organic phases were combined and sequentiallywashed with H₂O (100 mL) and brine. Drying over Na₂SO₄ and purificationon silica (hexanes/EtOAc, 1:1) afforded the title compound (17.46 g,96%) as a light yellow oil: MS (ES) m/e 401 (M+H)⁺.

b) 4-nitro-3-{[(N-methyl(phenylmethoxy)carbonylamino]methyl}benzoic acid

To a stirred solution oft-butyl-4-nitro-3-{[(N-methyl(phenylmethoxy)carbonylamino]methyl}benzoate(17.46 g, 43.65 mmole) in CH₂Cl₂ (100 mL) at RT was added TFA (50 mL).After 12 hr, the reaction contents were evaporated, washed with hexanesand dried under high vacuum overnight to afford the title compound(14.62 g, 42.5 mmol) as an orange oil. This product was used withoutfurther purification: MS (ES) m/e 345 (M+H)⁺.

c)N-[(2-nitro-5-{(N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-methyl(phenylmethoxy)carboxamide

To a stirred solution of4-nitro-3-{[(N-methyl(phenylmethoxy)carbonylamino]methyl}benzoic acid(4.56 g, 13.26 mmole) in DMF (20 mL) at RT was added diisopropylethylamine (2.31 mL, 13.26 mmole), HOBt (1.79 g, 13.26 mmole),1-methyl-2-(methylaminomethyl)indole (2.1 g, 12.0 mmole) and finally EDC(2.54 g, 13.26 mmole). After 12 hr, the reaction contents were pouredonto H₂O (100 mL) and extracted with EtOAc (2×100 mL). The organicphases were combined and sequentially washed with H₂O (100 mL) andbrine. Drying over Na₂SO₄ and purification on silica (hexanes/EtOAc,1:1) afforded the title compound (5.52 g, 92%) as a viscous yellow oil:MS (ES) m/e 501 (M+H)⁺.

d){4-amino-3-[(methylamino)methyl]phenyl}-N-methyl-N-[(1-methylindol-2-yl)methyl]carboxamide

To a solution ofN-[(2-nitro-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-methyl(phenylmethoxy)carboxamide(6.1 g, 12.2 mmole) in CH₃OH (50 mL) contained in a Parr hydrogenationflask was added a 0.75 g of 10% Pd/C. The contents were shaken on a Parrshaker under H₂ (50 psi) for 6 hours. The suspension was filteredthrough celite and concentrated under vacuum. Purification on silica[CHCl₃/CH₃OH (containing 5% NH₄OH), 9:1] afforded the title compound(3.40 g, 83%) as a viscous yellow oil: MS (ES) m/e 337 (M+H)⁺.

The following examples illustrate methods for preparing the biologicallyactive compounds of this invention from intermediate compounds such asthose described in the foregoing Preparations.

Example 1

Preparation ofN-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-phenylacetamide

a) tert-Butyl 3-[(phenylamino)methyl]-4-nitro benzoate

To a solution of the crude t-butyl-3-bromomethyl-4-nitro benzoate (2.0g, 6.3 mmole), from Procedure 3b, in THF (25 mL) at RT was added aniline(2.0 mL, 21.9 mmole). The reaction contents were concentrated, dissolvedin EtOAc and washed sequentially with 10% aqueous NaHCO₃ and brine.Purification on silica afforded the title compound (1.95 g, 94%) as anorange solid: MS (ES) m/e 429 (M+H)⁺.

b){3-[(phenylamino)methyl]phenyl-4-nitro}-N-methyl-N-[(1-methylindol-2-yl)methyl]carboxamide

To a stirred solution of tert-butyl 3-[(phenylamino)methyl]-4-nitrobenzoate (1.95 g, 4.55 mmole) in CHCl₂ (20 mL) at RT was added TFA (20mL). After 12 hr, the reaction contents were concentrated, treated with4M aqueous HCl, dioxane (10 mL) and concentrated under vacuum affordinga tan solid. The solid residue was washed with hexanes and dried underhigh vacuum. This product was used directly in the next step withoutfurther purification.

To a stirred solution of the above compound in DMF (30 mL) at RT wasadded diethyl amine (1.7 mL, 12.1 mmole),1-methyl-2-(methylaminomethyl)indole (1.0 g, 6.0 mmole), HOBt (0.81 g,6.0 mmole) and finally EDC (1.15 g, 6.0 mmole). After 12 hr, thereaction contents were poured onto H₂O (100 mL) and extracted with EtOAc(2×100 mL). The organic phases were combined and sequentially washedwith H₂O (100 mL) and brine. Drying over Na₂SO₄ and purification onsilica (EtOAc) afforded the title compound (2.33 g, 92%) as a solidyellow foam: MS (ES) m/e 429 (M+H)⁺.

c)N-[(2-nitro-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-phenylacetamide

To a solution of{3-[(phenylamino)methyl]phenyl-4-nitro}-N-methyl-N-[(1-methylindol-2-yl)methyl]carboxamide(0.75 g, 1.8 mmole) in CHCl₃ (10 mL) at 45° C. was added aceticanhydride (0.38 mL, 4.2 mmole) followed by pyridine (0.30 ml, 3.8mmole). After 12 hr, the reaction solution was concentrated undervacuum, dissolved in EtOAc and sequentially washed with 1 M HCl, andbrine. Drying over Na₂SO₄ and purification on silica (EtOAc) affordedthe title compound (0.82 g, 92%) as a yellow foam: MS (ES) m/e 493(M+Na)⁺.

d)N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-phenylacetamide

To a solution ofN-[(2-nitro-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-phenylacetamide(0.82 g, 1.7 mmole) in CH₃OH (50 mL) in a Parr hydrogenation flask wasadded 10% Pd/C (0.50 g). The contents were shaken on a Parr shaker underH₂ (50 psi) for 4 hours. The reaction suspension was filtered throughcelite and concentrated under vacuum. Purification on silica(CHCl₃/CH₃OH, 95:5) afforded the title compound (0.51 g, 68%) as anoff-white solid: MS (ES) m/e 441 (M+H)⁺.

Example 2

Preparation ofN-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-methylacetamide

To a stirred solution of 4-amino-3-[(N-methylacetylamino)methyl]benzoicacid trifluoro acetate (0.73 g, 2.17 mmole), from Procedure 3, in DMF(20 mL) at RT was added diisopropylethyl amine (0.83 ml, 4.78 mmole),HOBt (0.32 g, 2.39 mmole), 1-methyl-2-(methylaminomethyl)indole (0.40 g,2.39 mmole) and finally EDC (0.45 g, 2.39 mmole). After 12 hr, thereaction contents were poured onto H₂O (100 mL) and extracted with EtOAc(2×100 mL). The organic phases were combined and sequentially washedwith H₂O (100 mL) and brine. Drying over Na₂SO₄ and purification onsilica (CHCl₃/CH₃OH. 95:5) afforded the title compound (0.73 g, 89%) asa light yellow solid: MS (ES) m/e 379 (M+H)⁺.

Example 3

Preparation ofN-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-(2-phenylethyl)acetamide

According to the procedure of Example 2, except substituting4-amino-3-[(N-phenethylacetylamino)methyl]benzoic acid (0.60 g, 1.92mmole) for 4-amino-3-[(N-methylacetylamino)methyl]benzoic acid trifluoroacetate, the title compound (0.83 g, 92%) was prepared as a yellow solidfollowing chromatography on silica gel (CHCl₃/CH₃OH. 95:5): MS (ES) m/e469 (M+H)⁺.

Example 4

Preparation ofN-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-2-hydroxy-4-methyl-N-methylpentanamide

According to the procedure of Example 2, except substituting4-amino-3-[(2-hydroxy-4,N-dimethylpentanoylamino)methyl]benzoic acidtrifluoro acetate (2.1 g, 5.1 mmole) for4-amino-3-[(N-methylacetylamino)methyl]benzoic acid trifluoro acetate,the title compound (2.06 g, 90%) was prepared as a yellow foam followingchromatography on silica gel (CHCl₃/CH₃OH, 95:5): MS (ES) m/e 451(M+H)⁺.

Example 5

Preparation of{4-amino-3-[(ethoxy-N-methylcarbonylamino)methyl]phenyl}-N-methyl-N-[(1-methylindol-2-yl)methyl]carboxamide

According to the procedure of Example 2, except substituting4-amino-3-[(ethoxy-N-methylcarbonylamino)methyl]benzoic acid trifluoroacetate (0.75 g, 2.05 mmole) for4-amino-3-[(N-methylacetylamino)methyl]benzoic acid trifluoro acetate,the title compound (0.75 g, 90%) was prepared as a tan foam followingchromatography on silica gel (CHCl₃/CH₃OH, 95:5): MS (ES) m/e 409(M+H)⁺.

Example 6

Preparation ofN-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-2-hydroxy-N-methylacetamide

According to the procedure of Example 2, except substituting4-amino-3-[(2-hydroxy-N-methylacetylamino)methyl]benzoic acid (1.0 g,2.84 mmole) for 4-amino-3-[(N-methylacetylamino)methyl]benzoic acidtrifluoro acetate, the title compound (0.99 g, 88%) was prepared as anoff-white solid following chromatography on silica gel (CHCl₃/CH₃OH,95:5): MS (ES) m/e 395 (M+H)⁺.

Example 7

Preparation ofN-[(2-amino-5-{N-methyl-N-[(1-methylindol-3-yl)methyl]carbamoyl}phenyl)methyl]-N-methylacetamide

According to the procedure of Example 2, except substituting4-amino-3-[(N-methylacetylamino)methyl]benzoic acid trifluoroacetate(0.44 g, 1.31 mmole) for 4-amino-3-[(N-methylacetylamino)methyl]benzoicacid trifluoro acetate, the title compound (0.45 g, 92%) was prepared asan off-white solid following chromatography on silica gel (CHCl₃/CH₃OH,95:5): MS (ES) m/e 379 (M+H)⁺.

Example 8

Preparation ofN-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-2-hydroxy-3-indol-3-yl-N-methylpropanamide

According to the procedure of Example 2, except substituting4-amino-3-[(2-hydroxy-3-indol-3-yl-N-methylpropanoylamino)methyl]benzoicacid trifluoro acetate (0.41 g, 1.12 mmole) for4-amino-3-[(N-methylacetylamino)methyl]benzoic acid trifluoro acetate,the title compound (0.46 g, 78%) was prepared as an off-white solidfollowing chromatography on silica gel (CHCl₃/CH₃OH, 95:5): MS (ES) m/e524 (M+H)⁺.

Example 9

Preparation ofN-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-2-cyclopentyl-N-methylacetamide

According to the procedure of Example 2, except substituting4-amino-3-[(2-cyclopentyl-N-methylacetylamino)methyl]benzoic acidtrifluoro acetate (1.05 g, 3.60 mmole) for4-amino-3-[(N-methylacetylamino)methyl]benzoic acid trifluoro acetate,the title compound (1.45 g, 90%) was prepared as an off-white solidfollowing chromatography on silica gel (hexanes/EtOAc, 1:2): MS (ES) m/e448 (M+H)⁺.

Example 10

Preparation of{4-amino-3-{[(4-hydroxyphenyl)-N-methylcarbonylamino]methyl}phenyl}-N-methyl-N-[(1-methylindol-2-yl)methyl]carboxamide

According to the procedure of Example 2, except substituting4-hydroxybenzoic acid (0.23 g, 1.64 mmole) for4-amino-3-[(N-methylacetylamino)methyl]benzoic acid trifluoro acetateand substituting{4-amino-3-[(methylaminomethyl]phenyl}-N-methyl-N-[(1-methylindol-2-yl)methyl]carboxamide(0.50 g, 1.49 mmole) for 1-methyl-2-(methylaminomethyl)indole, the titlecompound (0.62 g, 92%) was prepared as an off-white solid followingchromatography on silica gel (CHCl₃/CH₃OH, 95:5): MS (ES) m/e 457(M+H)⁺.

Example 11

Preparation ofN-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-methyl-3-(phenylsulfonyl)propanamide

According to the procedure of Example 2, except substituting3-(phenylsulfonyl)propionic acid (0.35 g, 1.64 mmole) for4-amino-3-[(N-methylacetylamino)methyl]benzoic acid trifluoro acetateand substituting{4-amino-3-[(methylaminomethyl]phenyl}-N-methyl-N-[(1-methylindol-2-yl)methyl]carboxamide(0.50 g, 1.49 mmole) for 1-methyl-2-(methylaminomethyl)indole, the titlecompound (0.72 g, 91%) was prepared as an off-white solid followingchromatography on silica gel (CHCl₃/CH₃OH, 95:5): MS (ES) m/e 533(M+H)⁺.

Example 12

Parenteral Dosage Unit Composition

A preparation which contains 20 mg of the compound of Example 1 as asterile dry powder is prepared as follows: 20 mg of the compound isdissolved in 15 mL of distilled water. The solution is filtered understerile conditions into a 25 mL multi-dose ampoule and lyophilized. Thepowder is reconstituted by addition of 20 mL of 5% dextrose in water(D5W) for intravenous or intramuscular injection. The dosage is therebydetermined by the injection volume. Subsequent dilution may be made byaddition of a metered volume of this dosage unit to another volume ofD5W for injection, or a metered dose may be added to another mechanismfor dispensing the drug, as in a bottle or bag for IV drip infusion orother injection-infusion system.

Example 13

Oral Dosage Unit Composition

A capsule for oral administration is prepared by mixing and milling 50mg of the compound of Example 1 with 75 mg of lactose and 5 mg ofmagnesium stearate. The resulting powder is screened and filled into ahard gelatin capsule.

Example 14

Oral Dosage Unit Composition

A tablet for oral administration is prepared by mixing and granulating20 mg of sucrose, 150 mg of calcium sulfate dihydrate and 50 mg of thecompound of Example 1 with a 10% gelatin solution. The wet granules arescreened, dried, mixed with 10 mg starch, 5 mg talc and 3 mg stearicacid; and compressed into a tablet.

The above description fully discloses how to make and use the presentinvention. However, the present invention is not limited to theparticular embodiments described hereinabove, but includes allmodifications thereof within the scope of the following claims. Thevarious references to journals, patents and other publications which arecited herein comprises the state of the art and are incorporated hereinby reference as though fully set forth.

4 1 39 DNA Home sapiens 1 cgcctcgaga tgttaaatct tgaaaacaaa acatatgtc 392 33 DNA Homo sapiens 2 cgcggatcca atcaagtcag gttgaaatat cca 33 3 28 DNAHome sapiens 3 catgggctta aatcttgaaa acaaaaca 28 4 26 DNA Home sapiens 4tatgttttgt tttcaagatt taagcc 26

What is claimed is:
 1. A compound according to formula (I):

wherein: R¹ is C₁₋₄alkyl; R² is C₁₋₄alkyl; R³ is —C₁₋₄alkyl,—C₀₋₄alkyl-Ar or —C₀₋₄alkyl-Het, R⁴ is —C₁₋₄alkyl, —(CH₂)₁₋₄OH,—OC₁₋₄alkyl, —SC₁₋₄alkyl, —N(C₁₋₄alkyl)₂, —C₀₋₄alkyl-Ar, —C₀₋₄alkyl-Het,—C₀₋₄alkyl-C₃₋₆cycloalkyl, —CH(OH)—CH_(2-R*) or —(CH₂)₁₋₃SO₂Ar; R* isC₁₋₄alkyl, Ar or Het; X is H, C₁₋₄alkyl, OR′, SR′, CN, N(R′)₂,CH₂N(R′)₂, NO₂, CF₃, CO₂R′, CON(R′)₂, COR′, NR′C(O)R′, F, Cl, Br, I, or—S(O)_(r)CF₃; R′ is H, C₁₋₆alkyl or —C₀₋₆alkyl-Ar; and r is 0, 1 or 2;or a pharmaceutically acceptable salt thereof.
 2. A compound accordingto claim 1 of formula (Ia):


3. A compound according to claim 1 in which R³ is C₁₋₄alkyl or—C₀₋₂alkyl-Ph.
 4. A compound according to claim 1 in which R⁴ is—C₁₋₄alkyl, —CH₂OH, —OC₁₋₄alkyl, —C₀₋₂alkyl-Ph,—C₀₋₂alkyl-C₃₋₆cycloalkyl, —CH(OH)—CH₂—R* or —(CH₂)₂SO₂Ph.
 5. A compoundaccording to claim 1 which is:N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-methylacetamide;N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-(2-phenylethyl)acetamide;N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-2-hydroxy-4-methyl-N-methylpentanamide;{4-amino-3-[(ethoxy-N-methylcarbonylamino)methyl]phenyl}-N-methyl-N-[(1-methylindol-2-yl)methyl]carboxamide;N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-2-hydroxy-N-methylacetamide;N-[(2-amino-5-{N-methyl-N-[(1-methylindol-3-yl)methyl]carbamoyl}phenyl)methyl]-N-methylacetamide;N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-phenylacetamide:N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-2-hydroxy-3-indol-3-yl-N-methylpropanamide;(4-amino-3-{[(4-hydroxyphenyl)-N-methylcarbonylamino]methyl}phenyl)-N-methyl-N-[(1-methylindol-2-yl)methyl]carboxamide;N-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-N-methyl-3-(phenylsulfonyl)propanamide;orN-[(2-amino-5-{N-methyl-N-[(1-methylindol-2-yl)methyl]carbamoyl}phenyl)methyl]-2-cyclopentyl-N-methylacetamide;or a pharmaceutically acceptable salt thereof.
 6. A pharmaceuticalcomposition which comprises a compound according to claim 1 and apharmaceutically acceptable carrier.
 7. A method for inhibiting FabIwhich comprises administering to a subject in need thereof a compoundaccording to claim
 1. 8. A method of treating bacterial infections whichcomprises administering to a subject in need thereof a compoundaccording to claim
 1. 9. A process for preparing compounds of formula(I) as defined in claim 1, which process comprises reacting a compoundof formula (II) with a compound of formula (III):

wherein R¹, R², R³, R⁴ and X are as defined in formula (I), with anyreactive functional groups protected, in the presence of EDC and HOBT;and thereafter removing any protecting groups, and optionally forming apharmaceutically acceptable salt.